In 2019, we pursued studies that encompassed three major themes: 1) identification and characterization of T-bet-expressing B cells in lymph nodes of HIV-infected individuals with persistent HIV viremia; 2) role of IgG3 and other soluble factors in regulating B cells in HIV infection; and 3) studies on B cells in non-HIV diseases. First, in a large collaborative effort that has culminated in a manuscript accepted for publication in Science Translational Medicine, we completed a study identifying and characterizing a unique population of memory B cells (MBC) in lymph nodes of HIV-infected individuals. We began in 2015 with the aim of increasing our understanding of B-cell dysregulation that occurs in lymph nodes of HIV-infected individuals, especially those with chronic viremia. We employed two approaches, one based on advanced imaging of intact lymphoid tissue specimens and the other based on transcriptional and phenotypic profiling of cell suspensions obtained from the same tissue source. The two approaches eventually revealed a common theme: the transcription factor T-bet was expressed in a population of lymph node MBC in HIV-infected viremic individuals at levels that were much higher than in HIV-uninfected individuals. Given the strong polarizing effect of T-bet on T cells, we believed it could prove to have a similar effect on B cells; hence, our efforts to fully investigate this unique population of B cells that was over-represented in HIV-infected chronically-viremic individuals and associated with B-cell responses against the virus. We anticipated that these unusual B cells would be located in the germinal center (GC), where most of the HIV-induced lymphadenopathy has been observed. Instead, using advanced spatial and quantitative imaging that involved confocal microscopy and histo-cytometry, we observed that T-bet+ B cells accumulated outside the GC and expressed homing receptor and ligand profiles that were consistent with their non-GC positioning. Despite being physically excluded from the GC, phenotypic, functional, transcriptional and phylogenetic analyses revealed a close relationship between T-bet+ MBC and GC B cells, suggesting that one may be derived from the other. Furthermore, HIV-specific B cells were enriched among lymph node T-bet+ MBC and while both HIV-specific B cells and T-bet+ MBC harbored features of GC B cells, their accumulation outside the GC also translated into lower affinity maturation compared to GC B-cell counterparts. This is consistent with a paucity of T-cell help to B cells that remain outside the GC. Finally, HIV-specific monoclonal antibodies reconstituted with the specificity of respective B-cell receptors (BCR) exhibited lower levels of somatic hypermutation and HIV-neutralizing capacity when derived from MBC (enriched in T-bet+ B cells) as compared to those derived from GC B cells. We also found similarities between T-bet+ MBC in lymph nodes and the circulating tissue-like memory (TLM) B cells we first described in 2008 as being the source of HIV-associated B-cell exhaustion and later shown to also be deficient in affinity maturation. The current findings suggest that lymph node T-bet+ MBC may be the tissue source of TLM B cells that circulate in the peripheral blood of HIV-viremic individuals and in other infectious and non-infectious immune-activating conditions. Under the second major theme, we have continued to investigate the role of IgG3 and other soluble factors in regulating B cells in HIV infection. Last year we published findings describing a new phenomenon, unique to certain HIV-infected chronically viremic individuals, whereby soluble IgG3 binds to B cells through direct interactions with IgM that is expressed as part of the BCR of affected cells. Among B cells, the phenomenon is largely restricted to two subpopulations: nave B cells as well as TLM B cells that we first described over a decade ago as being over-expressed during chronic HIV viremia with features of HIV-associated B-cell exhaustion. Notably in the most recent findings, the binding of soluble IgG3 had a dampening effect on the function of TLM but not nave B cells. Mechanistically, we determined that soluble IgG3 bound to B cells in vivo through direct interactions with IgM in the BCR and that the binding of IgG3 involved three additional proteins: soluble C1q, the first protein of the complement cascade; soluble CRP, C-reactive protein of the acute-phase inflammatory response; and cell-surface expressed CD32b, the inhibitory Fc receptor for IgG. Together, these proteins assemble in vivo on B cells of HIV-infected individuals during chronic viremia and induce micro-clustering of the BCR, a process that normally occurs during antigen-specific BCR stimulation and signal transduction but that we found to restrict further activation in this context. Our current efforts have been aimed at delineating the kinetics of IgG3 binding to and clustering of the BCR during HIV disease and identifying factors in the serum of affected individuals that contribute to or modulate these events. Preliminary data on longitudinal specimens obtained from HIV-infected individuals undergoing analytical treatment interruption as part of clinical research protocols 16-I-0118 and 17-I-0106 are confirming and extending the observations that IgG3 binding to B cells increases during chronic HIV viremia and decreases when viremia is suppressed by antiretroviral therapy (ART). Under the third major theme, we have contributed to several collaborative studies on HIV and other diseases of the immune system with colleagues in the LIR, as well as with other laboratories at NIAID. In HIV-related studies, we collaborated with our colleagues in the LIR to investigate effects of various therapeutic agents on suppression of HIV in HIV-infected individuals and how certain proposed therapies may adversely affect bystander cells. In non-HIV studies, we used our expertise to assist several NIH colleagues in characterizing B cells in different settings. In one study published in the Journal of Experimental Medicine, we helped identify populations of B cells that were affected by the administration of glucocorticoids. In another study on systems biology that is under review in a high-impact journal, we helped characterize B-cell signatures that predict how the immune system responds to infection and other perturbations.